Agrobacterium tumefaciens twin-arginine-dependent translocation is important for virulence,flagellation, and chemotaxis but not type IV secretion

This study characterized the contribution of the twin-arginine translocation (TAT) pathway to growth, motility, and virulence of the phytopathogen Agrobacterium tumefaciens. In contrast to wild-type strain A348, a tatC null mutant failed to export the green fluorescent protein fused to the trimethylamine N-oxide reductase (TorA) signal sequence or to grow on nitrate as a sole electron acceptor during anaerobic growth. The tatC mutant displayed defects in growth rate and cell division but not in cell viability, and it also released abundant levels of several proteins into the culture supernatant when grown in rich medium or in vir induction minimal medium. Nearly all A348 cells were highly motile in both rich and minimal media. By contrast, approximately 0.1% of the tatC mutant cells were motile in rich medium, and <0.01% were motile in vir induction medium. Nonmotile tatC mutant cells lacked detectable flagella, whereas motile tatC mutant cells collected from the edge of a motility halo possessed flagella but not because of reversion to a functional TAT system. Motile tatC cells failed to exhibit chemotaxis toward sugars under aerobic conditions or towards nitrate under anaerobic conditions. The tatC mutant was highly attenuated for virulence, only occasionally (approximately 15% of inoculations) inciting formation of small tumors on plants after a prolonged incubation period of 6 to 8 weeks. However, an enriched subpopulation of motile tatC mutants exhibited enhanced virulence compared to the nonmotile variants. Finally, the tatC mutant transferred T-DNA and protein effectors to plant cells and a mobilizable IncQ plasmid to agrobacterial recipients at wild-type levels. Together, our findings establish that, in addition to its role in secretion of folded cofactor-bound enzymes functioning in alternative respiration, the TAT system of A. tumefaciens is an important virulence determinant. Furthermore, this secretion pathway contributes to flagellar biogenesis and chemotactic responses but not to sensory perception of plant signals or the assembly of a type IV secretion system.     

Loss of cytoskeletal transport during egress critically attenuates ectromelia virus infection in vivo

Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.     

Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.     

T cells from Leishmania major-susceptible BALB/c mice have a defect in efficiently up-regulating CXCR3 upon activation

Genetic background influences the outcome of Leishmania major infection. C57BL/6 mice mount a Th1 response and resolve infection. In contrast, BALB/c mice mount a Th2 response and develop chronic lesions. This susceptible phenotype is seen even though BALB/c mice generate IFN-gamma-producing T cells at proportions similar to C57BL/6 mice in their lymph nodes (LN) early after infection. We had previously shown that chemokine receptor CXCR3 mediates immunity against L. major by recruiting IFN-gamma-producing T cells to the lesions of C57BL/6 mice. Therefore, we hypothesized that IFN-gamma-secreting T cells in BALB/c mice are unable to confer protection because they may be defective in up-regulating CXCR3. To test this hypothesis, we analyzed kinetics of CXCR3-expressing T cells in the LN and lesions of BALB/c and C57BL/6 mice during L. major infection. Additionally, we compared the ability of T cells from BALB/c and C57BL/6 mice to up-regulate CXCR3 upon activation. We found that resolution of L. major infection in C57BL/6 mice was associated with an increase in the proportion of CXCR3(+) T cells in regional LN and lesions, whereas disease progression in BALB/c mice was associated with a decrease in these populations. Anti-CD3/CD28-activated T cells from naive BALB/c but not C57BL/6 mice were defective in up-regulating CXCR3. Impaired induction of CXCR3 on BALB/c T cells was not due to lack of IFN-gamma and was mediated partially by IL-10 but not IL-4 or IL-13. We propose that defective CXCR3 up-regulation on T cells in BALB/c mice may contribute to L. major susceptibility.     

Purification of the 110-kilodalton glycoprotein receptor for mouse hepatitis virus (MHV)-A59 from mouse liver and identification of a nonfunctional, homologous protein in MHV-resistant SJL/J mice.

The receptor for mouse hepatitis virus strain A59 (MHV-A59) is a 110- to 120-kilodalton (kDa) glycoprotein which is expressed in MHV-susceptible mouse strains on the membranes of hepatocytes, intestinal epithelial cells, and macrophages. SJL/J mice, which are highly resistant to MHV-A59, were previously shown to lack detectable levels of receptor by using either solid-phase virus receptor assays or binding of a monoclonal anti-receptor antibody (MAb) which blocks infection of MHV-susceptible mouse cells. This MAb was used for affinity purification of the receptor glycoprotein from livers of MHV-susceptible Swiss Webster mice. The MHV receptor and an antigenically related protein of 48 to 58 kDa were copurified and then separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 15 amino acids of the receptor were sequenced, and a synthetic peptide of this amino acid sequence was prepared. Rabbit antiserum made against this peptide bound to the MHV receptor glycoprotein and the 48- to 58-kDa protein from livers of MHV-susceptible BALB/c mice and Swiss Webster mice and from the intestinal brush border of BALB/c mice. In immunoblots of intestinal brush border and hepatocyte membranes of MHV-resistant SJL/J mice, the antibody against the amino terminus of the receptor identified proteins that are 5 to 10 kDa smaller than the MHV receptor and the 48- to 58-kDa related protein from Swiss Webster or BALB/c mice. Thus, SJL/J mice express a protein which shares some sequence homology with the MHV receptor but which lacks virus-binding activity and is not recognized by the blocking anti-receptor MAb. These results suggest that resistance of SJL/J mice to MHV-A59 may be due to absence or mutation of the virus-binding domain in the nonfunctional receptor homolog in SJL/J mice.     

Salmonella typhimurium infections in BALB/c mice: a comparison of tissue bioluminescence, tissue cultures and mice clinical scores

In response to systemic infection, mice usually present specific behaviors such as reduced activity and feeding, ruffled fur, hunched position, ataxia and tremor. We aimed to compare tissue bioluminescence, tissue cultures and clinical scores of BALB/c mice as potentially complementary outcome measures of Salmonella disease progression In Balb/c mice. The clinical status of the mice was assessed by visual examination for motility, ruffled fur, hunched position, feeding, ataxia and tremor. Patterns of bioluminescent light emission indicated the progression of infection from the abdominal region (initial site) to secondary tissue sites, which was indicative of systemic infection. As the severity and progression of infection increased, the bioluminescence signal became both more prominent and more anatomically disseminated. Bioluminescent Imaging (BLI) of Salmonella that have been genetically engineered to be bioluminescent is a new method that gives the opportunity to track Salmonella dissemination in mice. BLI is a helpful method to estimate tissue Salmonella concentration and may reduce the number of mice used in experiments, providing the opportunity to obtain serial assessments of disease progression in a single mouse subject. Clinical scores helped us to assess the clinical status of BALB/c mice in systemic Salmonella infections.     

IL-10 is required for prevention of necrosis in the small intestine and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice following peroral infection with Toxoplasma gondii

The role for IL-10 in the immunopathogenesis of acute toxoplasmosis following peroral infection was examined in both genetically susceptible C57BL/6 and resistant BALB/c mice. C57BL/6-background IL-10-targeted mutant (IL-10-/-) mice all died in 2 wk after infection with 20 cysts of the ME49 strain, whereas only 20% of control mice succumbed. Histological studies revealed necrosis in the small and large intestines and livers of infected IL-10-/- mice. The necrosis in the small intestine was the most severe pathologic response and was not observed in control mice. Treatment of infected IL-10-/- mice with either anti-CD4 or anti-IFN-gamma mAb prevented intestinal pathology and significantly prolonged time to death. Treatment of these animals with anti-IL-12 mAb also prevented the pathology. Significantly greater amounts of IFN-gamma mRNA were detected in the lamina propria lymphocytes obtained from the small intestine of infected IL-10-/- mice than those from infected control mice. In common with C57BL/6-background IL-10-/- mice, BALB/c-background IL-10-/- mice all died developing intestinal pathology after infection. Control BALB/c mice all survived even after infection with 100 cysts and did not develop the intestinal lesions. Treatment with anti-IFN-gamma mAb prevented the pathology and prolonged time to death of the infected IL-10-/- mice. These results strongly suggest that IL-10 plays a critical role in down-regulating IFN-gamma production in the small intestine following sublethal peroral infection with Toxoplasma gondii and that this down-regulatory effect of IL-10 is required for prevention of development of IFN-gamma-mediated intestinal pathology and mortality in both genetically resistant BALB/c and susceptible C57BL/6 mice.     

Characterization of Campylobacter jejuni RacRS reveals roles in the heat shock response, motility, and maintenance of cell length homogeneity

Campylobacter jejuni commensally colonizes the cecum of birds. The RacR (reduced ability to colonize) response regulator was previously shown to be important in avian colonization. To explore the means by which RacR and its cognate sensor kinase RacS may modulate C. jejuni physiology and colonization, ΔracR and ΔracS mutations were constructed in the invasive, virulent strain 81-176, and extensive phenotypic analyses were undertaken. Both the ΔracR and ΔracS mutants exhibited a ~100-fold defect in chick colonization despite no (ΔracS) or minimal (ΔracR) growth defects at 42 °C, the avian body temperature. Each mutant was defective for colony formation at 44°C and in the presence of 0.8% NaCl, both of which are stresses associated with the heat shock response. Promoter-reporter and real-time quantitative PCR (RT-qPCR) analyses revealed that RacR activates racRS and represses dnaJ. Although disregulation of several other heat shock genes was not observed at 38°C, the ΔracR and ΔracS mutants exhibited diminished upregulation of these genes upon a rapid temperature upshift. Furthermore, the ΔracR and ΔracS mutants displayed increased length heterogeneity during exponential growth, with a high proportion of filamented bacteria. Filamented bacteria had reduced swimming speed and were defective for invasion of Caco-2 epithelial cells. Soft-agar studies also revealed that the loss of racR or racS resulted in whole-population motility defects in viscous medium. These findings reveal new roles for RacRS in C. jejuni physiology, each of which is likely important during colonization of the avian host.     

A mutation in serca underlies motility dysfunction in accordion zebrafish

Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.     

Invariant NKT cell defects in vitamin D receptor knockout mice prevents experimental lung inflammation

Vitamin D receptor (VDR) deficiency (knockout [KO]) results in a failure of mice to generate an airway hyperreactivity (AHR) response on both the BALB/c and C57BL/6 background. The cause of the failed AHR response is the defective population of invariant NKT (iNKT) cells in the VDR KO mice because wild-type (WT) iNKT cells rescued the AHR response. VDR KO mice had significantly fewer iNKT cells and normal numbers of T cells in the spleen compared with WT mice. In BALB/c VDR KO mice, the reduced frequencies of iNKT cells were not apparent in the liver or thymus. VDR KO and WT Th2 cells produced similar levels of IFN-γ and IL-5. On the BALB/c background, Th2 cells from VDR KO mice produced less IL-13, whereas on the C57BL/6 background, Th2 cells from VDR KO mice produced less IL-4. Conversely, VDR KO iNKT cells were defective for the production of multiple cytokines (BALB/c: IL-4, IL-5, and IL-13; C57BL/6: IL-4 and IL-17). Despite relatively normal Th2 responses, BALB/c and C57BL/6 VDR KO mice failed to develop AHR responses. The defect in iNKT cells as a result of the VDR KO was more important than the highly susceptible Th2 background of the BALB/c mice. Defective iNKT cell responses in the absence of the VDR result in the failure to generate AHR responses in the lung. The implication of these mechanistic findings for human asthma requires further investigation.     

Calcineurin is required for Leishmania major stress response pathways and for virulence in the mammalian host

Leishmania parasites must adapt to elevated temperatures and other environmental stresses during infection of their mammalian hosts. How these environmental cues are sensed is poorly understood. In this study we show that calcium uptake is required for parasite thermotolerance at 34-37°C. To identify potential downstream targets of calcium influx, a Leishmania major mutant lacking the essential regulatory subunit (CnB) of the Ca(2+) /calmodulin-dependent serine/threonine-specific phosphatase, calcineurin, was generated. The Δcnb mutant grew as well as wild-type parasites at 27°C and differentiated normally to infective metacyclic promastigotes. However, Δcnb parasites lost viability when exposed to increased temperature (34°C) and were hypersensitive to endoplasmic reticulum and membrane stress, induced by tunicamycin and inhibitors of sterol and sphingolipid biosynthesis respectively. Δcnb promastigotes were internalized by macrophages, but their differentiation to the heat adapted amastigote stage was delayed and the resulting parasites failed to proliferate. Strikingly, the Δcnb parasites were completely cleared by susceptible BALB/c mice. Complementation of Δcnb parasites with CnB restored thermotolerance and infectivity in both macrophages and animal models. Our results suggest that Ca(2+) influx and calcineurin signalling are required for both early and long-term adaptive parasite responses to environmental stresses encountered in the mammalian host.     

Genetic resistance to mouse hepatitis virus correlates with absence of virus-binding activity on target tissues.

The molecular mechanism of genetic resistance of inbred mouse strains to mouse hepatitis virus, a murine coronavirus, was studied by comparing virus binding to plasma membranes of intestinal epithelium or liver from susceptible BALB/c and resistant SJL/J mice with a new solid-phase assay for virus-binding activity. Virus bound to isolated membranes from susceptible mice, but not to membranes from resistant mice. F1 progeny of SJL/J X BALB/c mice had an intermediate level of virus-binding activity on their enterocyte and hepatocyte membranes. This correlated well with previous studies showing that susceptibility to mouse hepatitis virus strain A59 is controlled by a single autosomal dominant gene (M. S. Smith, R. E. Click, and P. G. W. Plagemann, J. Immunol. 133:428-432). Because virus binding was not prevented by treating membranes with sodium dodecyl sulfate, the virus-binding molecule could be identified by a virus overlay protein blot assay. Virus bound to a single broad band of Mr 100,000 to 110,000 in membranes from hepatocytes or enterocytes of susceptible BALB/c and semisusceptible C3H mice, but no virus-binding band was detected in comparable preparations of resistant SJL/J mouse membranes. Therefore, SJL/J mice may be resistant to mouse hepatitis virus A59 infection because they lack a specific virus receptor which is present on the plasma membranes of target cells from genetically susceptible BALB/c and semisusceptible C3H mice.     

Mutations that impair swarming motility in Serratia marcescens 274 include but are not limited to those affecting chemotaxis or flagellar function.

Serratia marcescens exists in two cell forms and displays two kinds of motility depending on the type of growth surface encountered (L. Alberti and R. M. Harshey, J. Bacteriol. 172:4322-4328, 1990). In liquid medium, the bacteria are short rods with few flagella and show classical swimming behavior. Upon growth on a solid surface (0.7 to 0.85% agar), they differentiate into elongated, multinucleate, copiously flagellated forms that swarm over the agar surface. The flagella of swimmer and swarmer cells are composed of the same flagellin protein. We show in this study that disruption of hag, the gene encoding flagellin, abolishes both swimming and swarming motility. We have used transposon mini-Mu lac kan to isolate mutants of S. marcescens defective in both kinds of motility. Of the 155 mutants obtained, all Fla- mutants (lacking flagella) and Mot- mutants (paralyzed flagella) were defective for both swimming and swarming, as expected. All Che- mutants (chemotaxis defective) were also defective for swarming, suggesting that an intact chemotaxis system is essential for swarming. About one-third of the mutants were specifically affected only in swarming. Of this class, a large majority showed active "swarming motility" when viewed through the microscope (analogous to the active "swimming motility" of Che- mutants) but failed to show significant movement away from the site of initial inoculation on a macroscopic scale. These results suggest that bacteria swarming on a solid surface require many genes in addition to those required for chemotaxis and flagellar function, which extend the swarming movement outward. We also show in this study that nonflagellate S. marcescens is capable of spreading rapidly on low-agar media.     

Genetic control of lymphocyte suppression. I. Lack of suppression in aged NZB mice is due to a B cell defect.

Con A-activated cells from old NZB mice were found capable of inhibiting the polyclonal response of cells from young NZB and BALB/c animals. Furthermore, Con A-preactivated spleen cells from young NZB and BALB/c mice did not significantly affect the response of spleen cells from old NZB mice. These results suggest that the defective suppressive activity in old NZB mice may be traced to a defect at the B cell level.     

Induction of T15-specific suppressor T cells in mice with the CBA/N defect by neonatal injection with anti-T15 idiotype antibody

Mice with the CBA/N defect (xid) are unresponsive to phosphorylcholine (PC), To determine whether idiotype-specific suppressor T cells can also be generated in these defective mice, defective (CBA/N X BALB/c)F1 male and nondefective (CBA/N X BALB/c)F1 female or (BALB/c X CBA/N)F1 male mice were neonatally injected with antibodies specific for the major idiotype of anti-PC antibody, i.e., anti-TEPC-15 idiotype (T15id) antibody. Suppressor cell activity was examined by co-culturing spleen cells from neonatally treated F1 mice with spleen cells of normal nondefective F1 mice in the presence of antigen. Spleen cells from defective (CBA/NM X BALB/c)F1 mice treated with anti-T15id antibody demonstrated a level of suppressor activity (greater than 83% suppression) comparable to that of similarly treated nondefective F1 mice. This suppression was specific for the T15id of anti-PC response, and a Lyt-1-2+-bearing T cell population appeared to be responsible for the active suppression. These suppressor T cells recognized T15 but not PC, based on a functional absorption test. These results indicate that the CBA/N defects, including the deficiency in the anti-PC response by B lymphocytes and a possible T cell defect, do not influence the generation of T15id-specific suppressor T cells by neonatal injection with anti-T15id antibody.     

Catheter colonization and abscess formation due to Staphylococcus epidermidis with normal and small-colony-variant phenotype is mouse strain dependent

Coagulase-negative staphylococci (CoNS) form a thick, multilayered biofilm on foreign bodies and are a major cause of nosocomial implant-associated infections. Although foreign body infection models are well-established, limited in vivo data are available for CoNS with small-colony-variant (SCV) phenotype described as causative agents in implant-associated infections. Therefore, we investigated the impact of the Staphylococcus epidermidis phenotype on colonization of implanted PVC catheters and abscess formation in three different mouse strains. Following introduction of a catheter subcutaneously in each flank of 8- to 12-week-old inbred C57BL/6JCrl (B6J), outbred Crl:CD1(ICR) (CD-1), and inbred BALB/cAnNCrl (BALB/c) male mice, doses of S. epidermidis O-47 wild type, its hemB mutant with stable SCV phenotype, or its complemented mutant at concentrations of 10(6) to 10(9) colony forming units (CFUs) were gently spread onto each catheter. On day 7, mice were sacrificed and the size of the abscesses as well as bacterial colonization was determined. A total of 11,500 CFUs of the complemented mutant adhered to the catheter in BALB/c followed by 9,960 CFUs and 9,900 CFUs from S. epidermidis wild type in BALB/c and CD-1, respectively. SCV colonization was highest in CD-1 with 9,500 CFUs, whereas SCVs were not detected in B6J. The minimum dose that led to colonization or abscess formation in all mouse strains was 10(7) or 10(8) CFUs of the normal phenotype, respectively. A minimum dose of 10(8) or 10(9) CFU of the hemB mutant with stable SCV phenotype led to colonization only or abscess formation, respectively. The largest abscesses were detected in BALB/c inoculated with wild type bacteria or SCV (64 mm(2) vs. 28 mm(2)). Our results indicate that colonization and abscess formation by different phenotypes of S. epidermidis in a foreign body infection model is most effective in inbred BALB/c followed by outbred CD-1 and inbred B6J mice.     

Protection of mice from lethal endogenous Candida albicans infection by immunization with Candida membrane antigen

The protective effects of immunization with Candida membrane antigen (CMA) on a systemic infection originating from intestinally colonized Candida albicans were examined. The colonization of orally inoculated C. albicans in the intestinal tract was established in BALB/c mice that had been concomitantly treated with oral doses of antibacterial drugs. In these animals, a systemic dissemination of C. albicans with fatal outcome was induced by a repeated dosing of prednisolone. In this endogenous infection model, the effects of immunization by CMA on the infection were examined. CMA-immunized mice showed a longer lifespan than unimmunized mice. The protective effect of CMA immunization in immunosuppressed mice was also measured by a decrease in body weight loss after treatment with prednisolone and in the number of viable Candida cells in the target organs, the kidneys and livers. However, the CFU of C. albicans in the intestinal tract was not significantly lowered. These results suggest that CMA immunization inhibited the dissemination of systemic Candida infection from the intestinal tract induced by treatment with prednisolone.     

T cell-intrinsic factors contribute to the differential ability of CD8+ T cells to rapidly secrete IFN-γ in the absence of antigen

A subset of CD44(hi)CD8(+) T cells isolated from C57BL/6/J (B6) mice, but not BALB/c/By/J (BALB/c) mice, rapidly secrete IFN-γ within 16 h of infection with Listeria monocytogenes. This Ag-independent response requires the presence of both IL-12 and IL-18. Previous studies showed that dendritic cells from B6 mice produced more Th1-type cytokines such as IL-12 than did those from BALB/c mice in response to L. monocytogenes infection. In this report, we demonstrate that the microenvironment in L. monocytogenes-infected BALB/c mice is sufficient to induce responsive B6 CD8(+) T cells to rapidly secrete IFN-γ. Furthermore, BALB/c CD8(+) T cells did not rapidly secrete IFN-γ even when they were exposed to high concentrations of IL-12 plus IL-18 in vitro. In the presence of IL-12 and IL-18, B6 CD44(hi)CD8(+) T cells upregulated expression of the receptor subunits for these cytokines more rapidly than did BALB/c T cells. In comparing particular subsets of memory phenotype CD8(+) T cells, we found that virtual memory cells, rather than true Ag-experienced cells, had the greatest level of impairment in BALB/c mice. These data suggest that the degree of cytokine-driven bystander activation of CD8(+) T cells that occurs during infection depends on both APCs and T cell-intrinsic properties that can vary among mouse strains.